Follicle Oocyte Research Articles

Gonadal Atresia, Estrogen-Responsive, and Apoptosis-Specific mRNA Expression in Marine Mussels from the East China Coast: A Preliminary Study

This preliminary survey analysed mussel atresia incidences, estrogen-responsive and apoptotic-specific molecular end points, and aqueous and gonadal levels of selected estrogens from the East China coast. Estrogen levels were low (e.g. < LOD-28.36 ng/L, < LOD-3.88 ng/g wet weight of tissue for BPA) relative to worldwide freshwater environments, but high oocyte follicle atresia incidences (up to 26.6%) occurred at selected sites. Expression of estrogen-responsive ER2 was significantly increased in males relative to females at sites with high atresia incidences in females. A second estrogen-responsive gene, V9, was significantly increased at two sites in April in females relative to males; the opposite was true for the remaining two sites. Apoptosis-specific genes (Bcl-2, fas) showed elevated expression in males relative to females at the site with the highest atresia incidence. These results provide coastal estrogen levels and the utility of several estrogen-specific molecular-level markers for marine mussels.

Differential proteomic analysis demonstrates follicle fluid participate immune reaction and protein translation in yak

BackgroundOvarian follicle fluid (FF) as a microenvironment surrounding oocyte plays critical roles in physio-biochemical processes of follicle development and oocyte maturation. It is hypothesized that proteins in yak FF participate in the physio-biochemical pathways. The primary aims of this study were to find differentially expressed proteins (DEPs) between mature and immature FF, and to elucidating functions of the mature and immature FF in yak.ResultsThe mature and immature FF samples were obtained from three healthy yaks that were nonpregnant, aged from four to five years, and free from any anatomical reproductive disorders. The FF samples were subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ). The FF samples went through correlation analysis, principle component analysis, and expression pattern analysis based on quantification of the identified proteins. Four hundred sixty-three DEPs between mature and immature FF were identified. The DEPs between the mature and immature FF samples underwent gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) analysis. The DEPs highly expressed in the mature FF mainly took parts in the complement and coagulation cascades, defense response, acute-phase response, response to other organism pathways to avoid invasion of exogenous microorganisms. The complement activation pathway contains eight DEPs, namely C2, C5, C6, C7, C9, C4BPA, CFH, and MBL2. The three DEPs, CATHL4, CHGA, and PGLYRP1, take parts in defense response pathway to prevent invasion of exogenetic microorganism. The coagulation cascades pathway involves many coagulation factors, such as F7, F13A1, FGA, FGB, FGG, KLKB1, KNG1, MASP1, SERPINA1, and SERPIND1. While the DEPs highly expressed in the immature FF participated in protein translation, peptide biosynthetic process, DNA conformation change, and DNA geometric change pathways to facilitate follicle development. The translation pathway contains many ribosomal proteins, such as RPL3, RPL5, RPS3, RPS6, and other translation factors, such as EIF3J, EIF4G2, ETF1, MOV10, and NARS. The DNA conformation change and DNA geometric change involve nine DEPs, DDX1, G3BP1, HMGB1, HMGB2, HMGB3, MCM3, MCM5, MCM6, and RUVBL2. Furthermore, the expressed levels of the main DEPs, C2 and SERPIND1, were confirmed by western blot.ConclusionsThe differential proteomics revealed the up-regulated DEPs in mature FF take parts in immunoreaction to prevent invasion of microorganisms and the up-regulated DEPs in immature FF participate in protein synthesis, which may improve our knowledge of the follicular microenvironment and its biological roles for reproductive processes in yak. The DEPs, C2 and SERPIND1, can be considered as protein markers for mature yak follicle.

Oocyte CTR1 is not essential for cisplatin-induced oocyte death of primordial follicle.

Accumulated evidence indicates that cisplatin, a platinum-based alkylating agent, causes preferential DNA damage to oocytes of primordial follicles (PFs) in the ovary, suggesting oocyte-favored accumulation of cisplatin. Copper transporter 1 (CTR1; Slc31a1 ) is implicated in facilitating cisplatin uptake in cells. Here we found that oocytes of PFs had constitutively higher expression of CTR1 than other cell types in mouse ovary. However, oocyte-specific Slc31a1 knockout was not sufficient to prevent cisplatin-induced depletion of PFs in vitro . Our data indicate that CTR1 would not be the only route for cisplatin to be transported inside the oocytes of PFs in the ovary.

BCAS2 is involved in alternative splicing and mouse oocyte development.

Alternative splicing (AS) is an important mechanism to regulate organogenesis and fertility. Breast carcinoma amplified sequence 2 (BCAS2) is one of the core components of the PRP19 complex, a multiple function complex including splicing, and it is involved in the initiation of meiosis through regulating AS in male mice. However, the role of BCAS2 in mouse oogenesis remains largely unknown. In this study, we found that BCAS2 was highly expressed in the oocytes of primordial follicles. Vasa-Cre-mediated deletion of Bcas2 caused poor oocyte quality, abnormal oogenesis and follicular development. The deletion of Bcas2 in mouse oocytes caused alteration in 991 AS events that corresponded to 706 genes, including Pabpc1l, Nobox, Zfp207, Mybl2, Prc1, and Spc25, which were associated with oogenesis and spindle assembly. Moreover, the disruption of BCAS2 led to degradation of PRP19 core proteins in mouse oocytes. These results suggested that BCAS2 was involved in the AS of functional genes through PRP19 complex during mouse oocyte development.

Oocyte Survival and Development during Follicle Formation and Folliculogenesis in Mice Lacking Aromatase

ABSTRACT Background: Assembly of oocytes into primordial follicles is essential for establishing the ovarian reserve required for female fertility. In mice, this process begins during embryonic development. Primordial germ cells form cysts by incomplete mitosis until 13.5 days post coitum (dpc). These cysts break apart just before birth. Some oocytes undergo apoptosis while surviving oocytes are enclosed by granulosa cells to form primordial follicles. Cyst breakdown and primordial follicle formation were previously shown to be inhibited by estradiol and estrogenic compounds in vitro, suggesting that estrogen is important for regulation of this process. Methods: To determine the role of fetal estrogen in cyst breakdown and follicle formation these processes were quantified in aromatase deficient (ArKO) mice between 17.5 dpc and postnatal day (PND) 9. Ovaries of ArKO mice were also examined at 2-week intervals to determine if folliculogenesis is affected by lack of estrogen and the age at which the typical ArKO ovarian phenotype first appears. Results: Oocyte number, follicle assembly, and follicle development in ArKO mice did not differ from controls between 17.5 dpc and PND 9. At 2 weeks, ArKO ovaries still had oocytes in cysts while all oocytes were enclosed in follicles in wild type ovaries. From 2 to 8 weeks oocyte numbers were similar in all genotypes with a significant reduction at 10 weeks in ovaries from homozygous mutants. Abnormal hemorrhagic follicles were observed starting at 6 weeks, earlier than previously reported and hemosiderin deposits were found starting at 8 weeks. Conclusions: These results suggest that a lack of fetal estrogen does not affect oocyte survival or the rate of primordial follicle formation perinatally, and maternal estrogen or other signals are the chief regulators. The appearance of abnormal hemorrhagic follicles observed as early as 6 weeks suggests that the lack of estrogen becomes problematic at this time.

Induction of meiosis by embryonic gonadal somatic cells differentiated from pluripotent stem cells

BackgroundDepletion of oocytes leads to ovarian aging-associated infertility, endocrine disruption and related diseases. Excitingly, unlimited oocytes can be generated by differentiation of primordial germ cell like cells (PGCLCs) from pluripotent stem cells. Nevertheless, development of oocytes and follicles from PGCLCs relies on developmentally matched gonadal somatic cells, only available from E12.5 embryos in mice. It is therefore imperative to achieve an in vitro source of E12.5 gonadal somatic cells.MethodsWe explored to identify small molecules, which can induce female embryonic stem cells (ESCs) into gonadal somatic cell like cells.ResultsUsing RNA-sequencing, we identified signaling pathways highly upregulated in E12.5_gonadal somatic cells (E12.5_GSCs). Through searching for the activators of these pathways, we identified small-molecule compounds Vitamin C (Vc) and AM580 in combination (V580) for inducing differentiation of female embryonic stem cells (ESCs) into E12.5_GSC-like cells (E12.5_GSCLCs). After V580 treatment for 6 days and sorted by a surface marker CD63, the cell population yielded a transcriptome profile similar to that of E12.5_GSCs, which promoted meiosis progression and folliculogenesis of primordial germ cells. This approach will contribute to the study of germ cell and follicle development and oocyte production and have implications in potentially treating female infertility.ConclusionESCs can be induced into embryonic gonadal somatic cell like cells by small molecules.

ARE THERE OVARIAN RESPONSIVE INDEXES THAT PREDICT CUMULATIVE LIVE BIRTH RATES IN WOMEN OVER 39 YEARS?

Ovarian response indexes have been proposed in Assisted Reproductive Technology (ART) in order to optimize live birth rates (LBR), adjusting ovarian stimulation (OS) and minimizing risks. Gonadotropin doses are commonly adjusted according to ovarian reserve parameters, including antral follicle count (AFC), antimülerian hormone (AMH) and basal Follicle Stimulating Hormon (FSH) levels. The retrospective assessment of ovarian responses allows to identify three primary indexes: (i) Follicular output rate (FORT), the ratio of the number of pre-ovulatory follicles obtained at OS completion over AFC; (ii) Follicle oocyte index (FOI), the ratio of oocytes retrieved over AFC, and (iii) Ovarian sensitivity index (OSI), the ratio of oocytes retrieved over the total gonadotropin dose administered.

The effects of thirdhand smoke on reproductive health

Tobacco smoke is an environmental pollutant that can cause follicle destruction and oocyte dysfunction. Thirdhand smoke (THS) is residual tobacco smoke existing in the environment long after cigarettes have been extinguished, which can react with other environmental compounds to produce secondary pollutants. THS contains a variety of toxic and harmful chemicals, such as nicotine and 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA), a logical biomarker of THS exposure. The health hazards of THS exposure and its components have been researched in recent years. In this review, we have summarized research progress on the effects of THS exposure on organs in mice and humans especially on the reproductive system. This review may help evaluate the health risks of THS, in particular reproduction and offspring health. We hope this review will guide public health education on the dangers of THS exposure and promotion of healthy living habits.

P–678 Increased luteinizing hormone in ovarian dysfunction attenuates follicle development and oocyte quality in human

Abstract Study question Can increased luteinizing hormone impair follicular development and oocyte quality in patients with ovarian dysfunction? Summary answer Increased luteinizing hormone attenuates follicular development and oocyte quality, resulting in arrest of follicle growth and empty follicles and low-quality embryos. What is known already Patients with ovarian dysfunction exhibit elevated gonadotropins and low estrogen levels reflecting their low ovarian reserve. For ovarian stimulation in these patients, natural or mild stimulation protocols are likely used, but we often experienced the arrest of follicle growth and empty follicles at oocyte retrieval. Animal studies demonstrated that chronic high LH exposure impaired the growth of antral follicles by suppressing the expression of FSHR in granulosa cells via a modulation of intraovarian regulators, including the LH-induced thecal factors. Study design, size, duration: Retrospective analysis was conducted in 72 patients with ovarian dysfunction who received ovarian stimulations followed by IVF-ET from April 2018 to March 2020 after obtaining written informed consents under an approval from the ethical committee of our hospital. Participants/materials, setting, methods The data of hormonal levels, transvaginal ultrasound during ovarian stimulation and clinical outcome of IVF were extracted from electric chart. For evaluation of embryo, high quality embryos referred to embryos having Veeck classification &amp;gt;grade 3 and &amp;gt;4 blastomeres. Statistical significance was determined using Dunnett or chi-square tests, with P &amp;lt; 0.05 being statistically significant. Main results and the role of chance The median age of participants was 42 years of age (range 26–49) with low serum AMH levels (median 0.9 ng/ml, range 0–1.83). We analyzed 361 cycles of ovarian stimulation in total (median 4 cycles/patient, range 1–21). These stimulation cycles were classified into 3 groups; group A (n = 230): normal LH level, group B (n = 93): elevated LH level (&amp;gt; 10 mIU/ml) after ovarian stimulation and group C (n = 33): elevated LH level from the initiation of ovarian stimulation. Among 361 cycles, the arrest of follicle growth was detected in 5 cycles (group A: 0%, group B: 60%, group C: 40%). The proportions of empty follicle in group A, B and C were 17.3±2.0%, 20.9±3.3%and 38.6±7.2%, respectively. The rate of empty follicle was significantly high in group C. Although there was no significant difference in the rates of oocyte degeneration and fertilization, the rate of high-quality embryos in group C was 0.8-fold lower than that of group A. Limitations, reasons for caution Due to limitation of participants, we could not determine the appropriate LH level for ovarian stimulation in patients with ovarian dysfunction based on receiver operatorating characteristic curve. Wider implications of the findings: Normalization of LH levels for ovarian simulation in patients with ovarian dysfunction could improve follicle development and oocyte quality. Trial registration number Not applicable

P–416 Radiotherapy inflicts long-term damage upon the uterus, causing uterine artery endothelial dysfunction and pregnancy loss in mice

Abstract Study question Does the uterus sustain direct and long-term damage following radiotherapy, independent of ovarian damage? Summary answer Radiotherapy causes direct and long-term uterine damage. Ovariectomised and hormonally supplemented mice experience uterine artery endothelial dysfunction and pregnancy loss following transfer of healthy blastocysts. What is known already The detrimental off-target impacts of cancer therapies on the ovary are well established, with some fertility preservation techniques available to ensure patients maintain their fertility following gonadotoxic treatment. Low doses of radiotherapy kill the majority of primordial follicle oocytes in the ovary, reducing the ovarian reserve and fertile lifespan. Patients who have received radiotherapy experience higher rates of pregnancy complications including preterm birth, low birth weight, and stillbirth. However, no studies have investigated if radiation inflicts direct, fertility compromising damage to the uterus. Study design, size, duration Adolescent female mice were untreated or exposed to whole body y-irradiation (7Gy), then ovariectomised. Immediate damage was assessed up to 24 hours post-irradiation (n = 4/group). Four weeks following treatment, mice were hormone primed to induce endometrial receptivity (n = 7/group), artificial decidualisation (n = 7–8/group), or receive embryo transfers from healthy, unexposed donor mice to assess embryo implantation (n = 11–13/group), and mid-gestation development (n = 8–10/group). Participants/materials, setting, methods Four week old C57BL6/CBA (F1) female mice were used for this study. Immunofluorescence and in situ hybridisation were utilised to localise markers of immediate DNA damage and cell death following irradiation, and markers of receptivity in hormone primed uteri. Measurements of uterine artery blood flow were recorded using Doppler ultrasonography, and indices of pulsatility and resistance calculated. Uterine artery wire myography was performed to assess competency of endothelial and smooth muscle compartments following irradiation. Main results and the role of chance Within 24 hours of irradiation, DNA damage (yH2AX) and apoptosis (Puma/TUNEL) were elevated in uteri, compared to control. Irradiated mice that received embryo transfers from untreated donors had similar numbers of implantation sites 3-days post-transfer versus controls, however uteri were pale and atrophic suggesting impaired vascularisation. By 10-days post-transfer, implantation sites in irradiated mice were resorbing (p &amp;lt; 0.001), although uterine artery Doppler ultrasound measurements demonstrated no change in pulsatility or resistance indices. When the brain was shielded from irradiation to protect the hypothalamic-pituitary-gonadal axis, resorption still occurred (p &amp;lt; 0.001), suggesting direct uterine damage is the likely cause of pregnancy loss. To investigate uterine damage in the absence of an embryo, endometrial receptivity was induced artificially. Uteri from irradiated animals were lighter compared to control (p &amp;lt; 0.05), however localisation of receptivity markers (E-cadherin, Mucin1, Ki67) was normal. When decidualisation was artificially induced irradiated uteri were also lighter (p &amp;lt; 0.01) indicating impaired decidualisation and reduced capacity to adapt to pregnancy. Wire myography performed on uterine arteries demonstrated endothelial dysfunction in irradiated mice (p &amp;lt; 0.0001). Limitations, reasons for caution Here, only a single age and dose of radiotherapy exposure are modelled. Patients of all ages can receive many doses of radiotherapy in combination with various chemotherapies. Our highly manipulable model enables any treatment variation to be modelled and the effect on the uterus and pregnancy examined. Wider implications of the findings: Reproductive aged cancer patients need to be appropriately counselled regarding the risks to their long-term fertility following treatment. Until now, potential permanent impacts to the uterus following cancer treatment have not been considered. It is clear radiotherapy can impose long-term damage to the uterus and influence pregnancy success and fertility. Trial registration number NA

Distinct Signaling Pathways Distinguish in vivo From in vitro Growth in Murine Ovarian Follicle Activation and Maturation.

Women with cancer and low ovarian reserves face serious challenges in infertility treatment. Ovarian tissue cryopreservation is currently used for such patients to preserve fertility. One major challenge is the activation of dormant ovarian follicles, which is hampered by our limited biological understanding of molecular determinants that activate dormant follicles and help maintain healthy follicles during growth. Here, we investigated the transcriptomes of oocytes isolated from dormant (primordial) and activated (primary) follicles under in vivo and in vitro conditions. We compared the biological relevance of the initial molecular markers of mature metaphase II (MII) oocytes developed in vivo or in vitro. The expression levels of genes involved in the cell cycle, signal transduction, and Wnt signaling were highly enriched in oocytes from primary follicles and MII oocytes. Interestingly, we detected strong downregulation of the expression of genes involved in mitochondrial and reactive oxygen species (ROS) production in oocytes from primordial follicles, in contrast to oocytes from primary follicles and MII oocytes. Our results showed a dynamic pattern in mitochondrial and ROS production-related genes, emphasizing their important role(s) in primordial follicle activation and oocyte maturation. The transcriptome of MII oocytes showed a major divergence from that of oocytes of primordial and primary follicles.

Large lamellar bodies and their role in the growing oocytes of the armadillo Chaetophractus villosus.

Oogenesis in the armadillo Chaetophractus villosus, a representative species of a mammalian basal clade, was investigated by light microscopy, transmission electron microscopy, and immunohistochemical localization of keratin. At the beginning of the growth phase, oocyte follicles showed one, and sometimes several, large bodies composed of lamellae (multilamellar bodies [MLBs]), which entrap other cytoplasmic organelles at more advanced stages. Lamellae diameter is described in cross-section (37 nm) and tangential sections (50 nm). The MLB of early oocytes is most frequently located close to the nucleus. In large oocytes, both, this body and the free organelles are relocated at the oocyte periphery. The MLB grows from the primary follicle up to its full development at the follicular phase characterized by tall granulosa cells. Mitochondria, smooth small vesicles, and lipofuscin granules are trapped between lamellae. MLBs engage in the formation of different sets of organelles, both trapped and free ones. When oocytes are well developed and the zona pellucida is formed, the MLB is reduced to small remnants detected only by transmission electron microscopy. The MLB disintegrates when an antrum develops. Immunohistochemical localization techniques showed the presence of cytokeratin in the MLBs. This cytokeratin pool may be involved in the filament and desmosome formation found in the periphery of late oocytes.

Feasibility of Secondary Follicle Isolation, Culture and Achievement of In-Vitro Oocyte Maturation from Superovulated Ovaries: An Experimental Proof-of-Concept Study Using Mice.

Fertility preservation through ovarian stimulation, aiming at cryopreserving mature oocytes or embryos, is sometimes unsuccessful. This clinical situation deserves novel approaches to overcome infertility following cancer treatment in patients facing highly gonadotoxic treatment. In this controlled experimental study, we investigated the feasibility of in-vitro culturing secondary follicles isolated from superovulated ovaries of mice recently treated with gonadotropins. The follicle yields of superovulated ovaries were 45.9% less than in unstimulated controls. Follicles from superovulated ovaries showed faster growth pace during the initial 7 days of culture and secreted more 17β-estradiol by the end of culture vs controls. Parameters reflecting the outcome of follicular development and oocyte maturation competence in vitro were similar between superovulated and control groups, with a similar follicle size at the end of culture and around 70% survival. Nearly half of cultured follicles met the criteria for in-vitro maturation in both groups and approximately 60% of those achieved a mature MII oocyte, similarly in both groups. Over 60% of obtained MII oocytes displayed normal-looking spindle and chromosome configurations, without significant differences between the groups. Using a validated follicle culture system, we demonstrated the feasibility of secondary follicle isolation, in-vitro culture and oocyte maturation with normal spindle and chromosome configurations obtained from superovulated mice ovaries.

Differential Transcript Profiles in Cumulus-Oocyte Complexes Originating from Pre-Ovulatory Follicles of Varied Physiological Maturity in Beef Cows.

Small dominant follicle diameter at induced ovulation, but not at spontaneous ovulation, decreased pregnancy rate, fertilization rate, and day seven embryo quality in beef cows. We hypothesized that the physiological status of the follicle at GnRH-induced ovulation has a direct effect on the transcriptome of the Cumulus-Oocyte complex, thereby affecting oocyte competence and subsequent embryo development. The objective of this study was to determine if the transcriptome of oocytes and associated cumulus cells (CC) differed among small (≤11.7 mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7–14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). RNA sequencing data, from pools of four oocytes or their corresponding CC, revealed 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte pools from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. An additional 128, 98, and 80 DEG were identified among small versus large, small versus spontaneous, and large versus spontaneous follicle CC pools, respectively. The biological pathway “oxidative phosphorylation” was significantly enriched with DEG from small versus spontaneous follicle oocyte pools (FDR < 0.01); whereas the glycolytic pathway was significantly enriched with DEG from CC pools obtained from large versus small follicles (FDR < 0.01). These findings collectively suggest that altered carbohydrate metabolism within the Cumulus-Oocyte complex likely contributes to the decreased competency of oocytes from small pre-ovulatory follicles exposed to an exogenous GnRH-induced gonadotropin surge.

The Improvement and Clinical Application of Human Oocyte In Vitro Maturation (IVM).

Oocyte in vitro maturation (IVM) is a technology with a long history that was established before IVF. Although it has been studied extensively, the efficiency of IVM has been poor for almost 30 years. In terms of the benefits of IVM, the efficiency and adoption of IVM are being improved by some notable improvements that have occurred in recent years. The establishment of biphasic IVM is the most important advancement in recent years. Biphasic IVM includes the pre-IVM culturing phase and IVM phase. The CNP-mediated pre-IVM culturing system is specifically tailored for non/minimally stimulated immature oocytes, and its efficiency has been shown. This is the most significant improvement made in recent decades in this area. In the clinic, IVM can be used for PCOS patients to avoid the occurrence of ovarian hyperstimulation syndrome (OHSS). Additionally, this method can solve the reproductive problems of some patients with special diseases (resistant ovary syndrome) that cannot be solved by IVF. In most fertility preservation procedures, oocytes in small antral follicles are lost. However, IVM has the ability to capture this kind of oocyte and save reproductive potential. IVM can be easily combined with fertility preservation strategies that have been applied in the clinic and improve the efficiency of fertility preservation. IVM is a useful and attractive technology and may be used widely worldwide in the near future.

Coordinated Formation of IMPDH2 Cytoophidium in Mouse Oocytes and Granulosa Cells.

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme catalyzing de novo biosynthesis of guanine nucleotides, aggregates under certain circumstances into a type of non-membranous filamentous macrostructure termed “cytoophidium” or “rod and ring” in several types of cells. However, the biological significance and underlying mechanism of IMPDH assembling into cytoophidium remain elusive. In mouse ovaries, IMPDH is reported to be crucial for the maintenance of oocyte–follicle developmental synchrony by providing GTP substrate for granulosa cell natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system to produce cGMP for sustaining oocyte meiotic arrest. Oocytes and the associated somatic cells in the ovary hence render an exciting model system for exploring the functional significance of formation of IMPDH cytoophidium within the cell. We report here that IMPDH2 cytoophidium forms in vivo in the growing oocytes naturally and in vitro in the cumulus-enclosed oocytes treated with IMPDH inhibitor mycophenolic acid (MPA). Inhibition of IMPDH activity in oocytes and preimplantation embryos compromises oocyte meiotic and developmental competences and the development of embryos beyond the 4-cell stage, respectively. IMPDH cytoopidium also forms in vivo in the granulosa cells of the preovulatory follicles after the surge of luteinizing hormone (LH), which coincides with the resumption of oocyte meiosis and the reduction of IMPDH2 protein expression. In cultured COCs, MPA-treatment causes the simultaneous formation of IMPDH cytoopidium in cumulus cells and the resumption of meiosis in oocytes, which is mediated by the MTOR pathway and is prevented by guanosine supplementation. Therefore, our results indicate that cytoophidia do form in the oocytes and granulosa cells at particular stages of development, which may contribute to the oocyte acquisition of meiotic and developmental competences and the induction of meiosis re-initiation by the LH surge, respectively.

Figla promotes secondary follicle growth in mature mice

The in vitro growth (IVG) of human follicles is a potential fertility option for women for whom cryopreserved ovarian tissues cannot be transplanted due to the risk of cancer cell reintroduction; however, there is currently no established method. Furthermore, optimal IVG conditions may differ between the follicles of adult and pre-pubertal females due to molecular differences suggested by basic research. To systematically identify differences between the secondary follicles of adult and pre-pubertal females, a comparative transcriptomic study using mice was conducted herein. Among differentially expressed genes (DEGs), Figla was up-regulated in mature mice. We successfully down-regulated Figla expression in secondary follicle oocytes by a Figla siRNA microinjection, and the subsequent IVG of follicles showed that the diameter of these follicles was smaller than those of controls in mature mice, whereas no significant difference was observed in premature mice. The canonical pathways of DEGs between control and Figla-reduced secondary follicles suggest that Figla up-regulates VDR/RXR activation and down-regulates stem cell pluripotency as well as estrogen signaling. We demonstrated for the first time that folliculogenesis of the secondary follicles of premature and mature mice may be regulated by different factors, such as Figla with its possible target genes, providing insights into optimal IVG conditions for adult and pre-pubertal females, respectively.

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

DNA repair in primordial follicle oocytes following cisplatin treatment.

Genotoxic chemotherapy and radiotherapy can cause DNA double stranded breaks (DSBs) in primordial follicle (PMF) oocytes, which then undergo apoptosis. The development of effective new fertility preservation agents has been hampered, in part, by a limited understanding of DNA repair in PMF oocytes. This study investigated the induction of classical DSB repair pathways in the follicles of wild type (WT) and apoptosis-deficient Puma-/- mice in response to DSBs caused by the chemotherapy agent cisplatin. Adult C57BL/6 WT and Puma-/- mice were injected i.p. with saline or cisplatin (5 mg/kg); ovaries were harvested at 8 or 24 h. Follicles were counted, and H2A histone family member (γH2AX) immunofluorescence used to demonstrate DSBs. DNA repair protein RAD51 homolog 1 (RAD51) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) immunofluorescence were used to identify DNA repair pathways utilised. Puma-/- mice retained 100% of follicles 24 h after cisplatin treatment. Eight hours post-treatment, γH2AX immunofluorescence showed DSBs across follicular stages in Puma-/- mice; staining returned to control levels in PMFs within 5 days, suggesting repair of PMF oocytes in this window. RAD51 immunofluorescence eight hours post-cisplatin was positive in damaged cell types in both WT and Puma-/- mice, demonstrating induction of the homologous recombination pathway. In contrast, DNA-PKcs staining were rarely observed in PMFs, indicating non-homologous end joining plays an insignificant role. PMF oocytes are able to conduct high-fidelity repair of DNA damage accumulated during chemotherapy. Therefore, apoptosis inhibition presents a viable strategy for fertility preservation in women undergoing treatment.